The transcriptional machinery of HIV-1 remains a therapeutic target that has not been exploited in current therapeutic regimens. HIV transcription is uniquely dependent on the action of the viral transactivator Tat. In addition, integration into the human genome and organization into chromatin is a key regulatory step for HIV-1 transcription. Reversible post-translational modifications of core histones, the subunits of chromatin, are known mechanisms to control chromatin organization and gene activation. We have obtained evidence that phosphorylation of histone H3 in the HIV promoter is increased in the presence of Tat. Phosphorylation of histone H3 is an early marker of gene activation and is mediated by the p90 ribosomal S6 kinase 2 (RSK2). Expression of a dominant-negative RSK2 enzyme inhibited Tat-mediated transactivation of the HIV-1 promoter, indicating that RSK2 is an important cellular factor for Tat function. In contrary, introduction of an intact RSK2 gene into human fibroblasts derived from a patient lacking RSK2 enhanced Tat transactivation in accordance with the model that RSK2 supports Tat function. Tat and RSK2 physically interact in cells, and interestingly, RSK2 activity is markedly increased in the presence of Tat. In this proposal we plan to further study the Tat/RSK2 interaction in the context of HIV infection. We will identify the domains in both proteins that are critical for protein/protein interaction and will generate binding-deficient point mutants of Tat and RSK2 to study their effect on the HIV promoter. We will analyze the molecular mechanisms of Tat-induced RSK2 activation by performing extensive phosphomapping studies of the endogenous RSK2 protein in the presence of Tat. We will also make use of existing phospho-RSK2 antibodies and will generate, if necessary, new antibodies against specific Tat-induced phosphorylation sites in RSK2. To extend our observations to HIV infection, we will infect human RSK2-deficient fibroblasts with pseudotyped HIV-based lentiviral vectors. In addition, we will generate T cell lines in which RSK2 has been stably knocked down by siRNA technology and study replication of infectious HIV in these cells. We anticipate that these studies will substantially further our understanding of H/V pathogenesis and explore its potential as a novel target for anti-HIV therapy.